Parent Grant Abstract/Summary Cells migrating in tissues use filopodia to interact with and move through their 3D environment. Filopodia are slender actin-filled projections composed of a core of cross-linked, parallel actin bundles. They are highly dynamic, vary in length and are found in a wide variety of cell types such as neurons that use them for gradient sensing and efficient directional migration and cancer cells that employ them for invading into neighboring tissue. The mechanism of filopodia initiation is not well understood. Three conserved proteins are required for their formation - a MyTH4-FERM myosin (MF; MyTH4 = myosin tail homology 4; FERM = band 4.1, ezrin, radixin, moesin) and two regulators of actin polymerization, VASP and Formin. How the action of these three proteins is coordinated to generate filopodia is unknown. The objective of the parent project is to define the molecular mechanism of filopodia initiation with an emphasis on the role of a MF myosin in this process. The versatile model system, Dictyostelium will be used to define how a MF myosin and VASP work together to organize the fast growing ends of actin filaments at the membrane to initiate construction of an actively extending actin filament bundle. A combination of in vivo, in vitro and in silico approaches are proposed to a) determine the functional relationship between a MF myosin, VASP and formin, b) identify the specific properties of the myosin motor used for filopodia initiation, c) identify proteins that interact with the MF myosin to promote cortical targeting during filopod initiation and filopod tip formation and d) develop a stochastic computational model with predictive power that will inform the experimental goals, the results of which will be used to refine the model. The knowledge generated from the parent project will reveal how cells use a myosin-based motor to build specific actin-based structures such as filopodia. Understanding how initiation occurs will also reveal how cells control filopodia formation to enable directed migration or invasion of surrounding tissues.